CN108700519B

CN108700519B - Methods and compositions for fluorescence detection

Info

Publication number: CN108700519B
Application number: CN201780013075.5A
Authority: CN
Inventors: C·T·麦基
Original assignee: Bioradiation Laboratory Co ltd
Current assignee: Bioradiation Laboratory Co ltd
Priority date: 2016-02-24
Filing date: 2017-02-23
Publication date: 2021-07-30
Anticipated expiration: 2037-02-23
Also published as: EP3420341A1; WO2017147267A1; CN108700519A; US20170242002A1; US10605807B2; EP3420341B1; EP3420341A4

Abstract

Disclosed herein are methods and compositions for imaging and scanning Western or immunoblotting. The composition comprises a film immersed in a solution comprising 70% w/v or more of a sugar and a zwitterionic surfactant, wherein the sugar and zwitterionic surfactant are dissolved in water or an aqueous buffer; and the membrane is configured for use in an analyte detection assay. A method of imaging a membrane in an analyte detection assay comprising: immersing the membrane in the solution, and imaging the membrane. Also disclosed is a kit for detecting an analyte, the kit comprising the solution and a membrane configured for use in an analyte detection assay.

Description

Methods and compositions for fluorescence detection

Cross Reference to Related Applications

This application claims the benefit of U.S. provisional application No. 62/299,436 filed on 24/2/2016, which is incorporated herein in its entirety for all purposes.

Background

Western or dot blot is commonly used as an analytical technique to detect various proteins. Fluorescence detection is one of the methods of choice in most Western and dot blot assays for visualization and quantification of proteins. However, nitrocellulose, polyvinylidene fluoride (PVDF), or other membranes commonly used for assays scatter incident light, making protein quantification difficult. Accordingly, there is a need in the art to provide novel methods and compositions to address this problem of light scattering.

Disclosure of Invention

Broadly, provided herein are methods for detecting and/or quantifying the fluorescent signal of nitrocellulose, PVDF, or other suitable membranes used in western blotting or other analytical techniques. The inventors have found that an aqueous mixture of a sugar and a surfactant provides a solution having the following characteristics: it reduces the background signal of standard capture membranes (e.g., PVDF, nitrocellulose) and increases the signal of fluorescent detection reagents. This solution (which may also include a buffer) may be used as the final membrane incubation solution, for example, in a Western blot detection workflow, to improve detection of the target protein.

One provided composition comprises 70% weight/volume (w/v) or more of a sugar, and a zwitterionic surfactant. The sugar and zwitterionic surfactant are dissolved in water or an aqueous buffer. In some embodiments, the zwitterionic surfactant of the composition is an aminothiobetaine. In some embodiments, the aminothiobetaine is 3- (dimethyl (3- (4-octylbenzamido) propyl) ammonio) propane-1-sulfonate (ASB-C80). In some embodiments, the zwitterionic surfactant concentration is in a range of about 0.01% to about 0.2% volume/volume (v/v). In some embodiments, the saccharide in the composition is selected from the group consisting of: sucrose, glucose, maltose, lactose, dextrose, cellobiose, and galactose. In some embodiments, the concentration of sugar in the composition is in the range of about 70% to about 90% w/v. In some embodiments, the composition further comprises a membrane immersed in water or an aqueous buffer, wherein the membrane is configured for use in an analyte detection assay. In some embodiments, the membrane of the composition comprises nitrocellulose or PVDF. In some embodiments, the analyte detection assay is a Western blot.

Also provided is a method of imaging a membrane in an analyte detection assay, the method comprising: the membrane was immersed in the solution. The membrane is configured for use in an analyte detection assay. The solution comprises a sugar and a zwitterionic surfactant, wherein the sugar and zwitterionic surfactant are dissolved in water or an aqueous buffer. The method further comprises the following steps: the film is imaged. In some embodiments, the membrane of the method comprises nitrocellulose or PVDF. In some embodiments, the analyte detection assay is a Western blot. In some embodiments, the zwitterionic surfactant of the method is an aminothiobetaine. In some embodiments, the aminothiobetaine of the method is 3- (dimethyl (3- (4-octylbenzamido) propyl) ammonio) propane-1-sulfonate (ASB-C80). In some embodiments, the zwitterionic surfactant concentration in the method is in the range of about 0.01% to about 0.2% v/v. In some embodiments, the saccharide in the method is selected from the group consisting of: sucrose, glucose, maltose, lactose, dextrose, cellobiose, and galactose. In some embodiments, the concentration of sugar in the method is in the range of about 70% to about 90% w/v.

Also provided is a kit for detecting an analyte, the kit comprising a saccharide, a zwitterionic surfactant, and a buffer. In some embodiments, the zwitterionic surfactant of the kit is aminothiobetaine. In some embodiments, the aminothiobetaine of the kit is 3- (dimethyl (3- (4-octylbenzamido) propyl) ammonio) propane-1-sulfonate (ASB-C80). In some embodiments, the saccharide in the kit is selected from the group consisting of: sucrose, glucose, maltose, lactose, dextrose, cellobiose, and galactose. In some embodiments, the kit further comprises a binding agent.

Drawings

FIG. 1 is a photograph of a polyvinylidene fluoride (PVDF) membrane immersed in 85% w/v sugar solution or water.

FIG. 2 is a graph showing the effect of sugar and surfactant on Westen blot emission quenching.

FIG. 3 is a graph showing the effect of surfactant on Westen blot emission intensity.

Figure 4 presents a graph showing the effect of sugar and surfactant on western blot emission intensity at different protein concentrations.

Fig. 5 presents a photograph of a membrane showing the improved analyte resolution when immersed in a sugar/surfactant solution.

Fig. 6 presents photographs and graphs showing the improvement in analyte resolution and emission intensity when immersed in a sugar/surfactant solution.

FIG. 7 is a graph showing the effect of different surfactants on Westen blot emission quenching.

FIG. 8 is a graph showing the effect of surfactant concentration on Westen blot emission quenching.

Definition of

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. See, e.g., Lackie, Dictionary of Cell and Molecular Biology, Elsevier (Elsevier) (4 th edition, 2007); and Sambrook et al, Molecular Cloning, A Laboratory Manual, Cold spring harbor Laboratory Press (Cold spring harbor, New York 1989 and subsequent). The terms "a" or "an" are intended to mean "one or more". The word "comprising" and variations thereof, such as "comprises" and "comprising," when used in conjunction with a stated step or element, is intended to indicate that the addition of another step or element is optional and non-exclusive. Methods, devices, and materials similar or equivalent to those described herein can be used in the practice of the present invention. The following definitions are provided to aid in the understanding of certain terms used frequently herein and are not meant to limit the scope of the present invention. The abbreviations used herein have their conventional meaning in the chemical and biological arts.

As used herein, the term "saccharide" refers to a monosaccharide, disaccharide, oligosaccharide or polysaccharide. Monosaccharides include, but are not limited to, glucose, ribose, fructose, mannose, xylose, arabinose, and galactose. Disaccharides include, but are not limited to: sucrose, lactose, cellobiose and maltose. Polysaccharides include, but are not limited to: cellulose, hemicellulose, and lignocellulose or starch. Other sugars may be used in the present invention.

The term "surfactant" as used herein refers to a surface active agent (surfactant) used to reduce the surface tension between liquids or between liquids and solids. Surfactants may be used as detergents, wetting agents, emulsifiers, foaming agents or dispersing agents.

The term "zwitterionic surfactant" as used herein refers to an amphiphilic surfactant molecule having no net charge, which comprises a hydrophobic group and one or more hydrophilic groups, and two moieties of opposite formal charge.

The term "buffer" as used herein refers to any inorganic or organic acid or base that resists changes in pH and maintains the pH of the solution to which it is added near a desired point.

The term "membrane" as used herein refers to a sheet of polymeric material which is used to have an analyte detectable by suitable chemical means exposed on its surface. Analytes can be transferred to the membrane by electroblotting from an electrophoresis gel.

The terms "analyte", "antigen" and "target" as used herein refer to any molecule, compound or complex of interest, the presence, amount, level of expression, state of activation and/or species of which is determined. This can be determined by specific recognition of the binding agent. The molecule, compound, or complex of interest can be a macromolecule, such as, for example, a polypeptide or protein, polysaccharide, toxin, cell wall, cell capsule, viral coat, flagellum, cilia or pilus, microorganism, nucleic acid complexed to a protein or polysaccharide, lipid complexed to a protein or polysaccharide, polynucleotide, polypeptide, carbohydrate, chemical moiety, or a combination thereof (e.g., phosphorylated or glycosylated polypeptide, etc.). It will be understood by those skilled in the art that the term does not indicate that the analyte is immunogenic in every instance, but merely that it can be targeted by a binding agent or antibody.

The term "binding agent" as used herein refers to a molecule that specifically binds an antigen or analyte. Exemplary binding agents include, but are not limited to, antibodies, antibody fragments, non-antibody protein scaffolds, antibody mimetics, aptamers, affimers, quenchers (quechbodies), enzyme-labeled antibodies, or analyte-specific antibody pairs.

The term "binding" as used herein with respect to a binder and a target means that the binder is attached to the majority of the target in the pure population (assuming an appropriate molar ratio of binder to target). For example, a binding agent that binds to a given target will bind at least 2/3 of the target in solution (e.g., 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%). One skilled in the art will recognize that some variability will arise depending on the affinity of the binding agent for the target and the method and/or threshold for determining binding.

The term "specifically binds" or "specific for …" as used herein refers to a molecule (e.g., a binding agent) that has at least 2-fold greater binding affinity for a target than a non-target compound, e.g., at least 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 25-fold, 50-fold, or 100-fold greater binding affinity. When one or more detectable protein analytes are used, specific binding is a determining factor for the presence of proteins in other biological agents and heterogeneous populations of proteins. Thus, under a given immunoassay condition, a particular antibody binds to a particular protein sequence, thereby identifying its presence.

Herein, the term "antibody" refers to a polypeptide of the immunoglobulin family or a polypeptide comprising an immunoglobulin fragment capable of binding a corresponding epitope noncovalently, reversibly and in a specific manner. The term includes, but is not limited to: polyclonal or monoclonal antibodies of the IgA, IgD, IgE, IgG and IgM isotype class derived from human or other mammalian cells include natural or genetically modified forms such as humanized, human, single chain, chimeric, synthetic, recombinant, hybrid, mutated, grafted and in vitro generated antibodies. The term includes conjugates, including, but not limited to, fusion proteins containing immunoglobulin moieties (e.g., chimeric or bispecific antibodies or single chain Fv's (scFv's)) and fragments (e.g., Fab, F (ab ')2, Fv, scFv, Fd, dAb, and other compositions).

The term "epitope" as used herein refers to a local site on an antigen that is recognized and bound by an antibody. A protein epitope may comprise several amino acids or parts of several amino acids, for example, 5 or 6 or more, or 20 or more amino acids or parts of these amino acids. Epitopes can also include non-protein components, such as nucleic acids (e.g., RNA or DNA), carbohydrates, lipids, or combinations thereof. The epitope may be a three-dimensional portion. Thus, for example, where the target is a protein target, an epitope can include amino acids that are contiguous, or from different portions of the protein that are adjacent by folding of the protein (e.g., a discontinuous epitope). This is also true for other types of target molecules that form three-dimensional structures (e.g., DNA and chromatin).

The terms "label" and "detectable label" as used herein refer to a composition that is detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical or other physical means. Useful labels include fluorescent dyes (fluorophores), fluorescence quenchers, luminescent agents, high electron density reagents, enzymes (e.g., enzymes commonly used in ELISA), biotin, digoxigenin, fluorescent dyes, high electron density reagents, enzymes, e.g., enzymes commonly used in ELISA, fluorescent dyes, high electron density reagents, enzymes, and the like,³²P and other radioisotopes, haptens, proteins, nucleic acids or other substances that can be detected, for example by incorporating or attaching labels to antibodies, peptides or oligonucleotides that specifically react with the target molecule. The term includes combinations of single labeling agents, e.g., combinations of fluorophores that provide a unique detectable characteristic (e.g., at a particular wavelength or combination of wavelengths).

As used herein, the term "linked" with respect to a label and a binding agent (e.g., a labeled antibody as described herein) means that the bound label is bound to the binding agent covalently (by a linker or chemical bond) or noncovalently (by ionic, van der waals, electrostatic, or hydrogen bonding) such that the presence or absence of the analyte can be detected by determining the presence or absence of the label bound to the binding agent.

The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. All three terms apply to amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of the corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. As used herein, the term encompasses amino acid chains of any length, including full length proteins, wherein the amino acid residues are linked by covalent peptide bonds.

As used herein, the term "Western blotting" or "Western immunoblotting" refers to analytical techniques for detecting a particular protein in a biological or other sample of interest. The technique may use gel electrophoresis or other suitable processes to separate proteins by size, shape, length, charge, or other characteristics. The proteins may then be transferred to a membrane and detected with a binding agent that may be specific for one or more proteins of interest.

The terms "about" and "approximately" are used herein to modify a numerical value and to represent the range defined around that value. If "X" is a value, "about X" or "approximately equal to X" typically represents a value of 0.90X to 1.10X. Any reference to "about X" means at least the values X, 0.90X, 0.91X, 0.92X, 0.93X, 0.94X, 0.95X, 0.96X, 0.97X, 0.98X, 0.99X, 1.01X, 1.02X, 1.03X, 1.04X, 1.05X, 1.06X, 1.07X, 1.08X, 1.09X, and 1.10X. Thus, "about X" is also intended to disclose, for example, "0.98X". When "about" is used at the beginning of a range of values, it is used at both ends of the range. Thus, "about 6 to 8.5" is equivalent to "about 6 to about 8.5". When "about" is used for the first value of a set of values, it is used for all values in the set. Thus, "about 7, 9, or 11%" equals "about 7%, about 8%, or about 11%".

Detailed Description

I. Overview

Western blots were first described in 1979 by Towbin et al ((1979) Proc. Natl. Acad. Sci USA 76: 4350) and have been used for protein detection and quantification using antibodies or other detection means. Early applications of this technology relied on antibody-enzyme conjugates (including horseradish peroxidase or alkaline phosphatase), and were only semi-quantitative. Later protocols utilized the development of fluorescent and chemiluminescent dye tags that, for example, provided more quantitative determination of proteins. However, the inherent autofluorescence of membranes associated with these techniques remains an unresolved problem.

Described herein is a novel method for reducing autofluorescence of membranes for Western blotting and other analytical processes, and increasing the fluorescence intensity of fluorophore labels for detection and quantification of target analytes. The present invention provides compositions and methods comprising specific solutions of a sugar and a surfactant. The inventors have found that solutions with a very concentrated concentration range of sugars minimize the effects of autofluorescence and light scattering when imaging membranes immersed therein. The inventors have also found that the addition of a surfactant to the sugar solution serves to at least partially mitigate quenching of the desired fluorescence emission associated with the detection of the relevant target analyte.

Composition II

Various compositions are provided herein. The compositions can be used as imaging solutions for analyte detection assays. The analyte detection assay may be a Western blot. It has been observed that the addition of a sugar to the composition can result in reduced autofluorescence or light scattering upon imaging of a film immersed in the composition.

The sugar of the composition may be any sugar that is highly water soluble. For example, sucrose, fructose, glucose, maltose, dextrose, or lactose may be used. Other monosaccharides, disaccharides, and polysaccharides may also be used. For example, the saccharide can be a monosaccharide, e.g., xylose, fucose, tagatose, galactosamine, glucosamine, mannosamine, galactose, mannose, galacturonic acid, glucuronic acid, iduronic acid, mannuronic acid, N-acetylgalactosamine, N-acetylglucosamine, N-acetylmannosamine, N-acetylmuramic acid, 2-keto-3-deoxy-glycero-galactosyl-nonanoic acid, N-acetylneuraminic acid or N-neuraminic acid. The sugar may be a disaccharide, for example lactulose, trehalose, cellobiose, isomaltose, isomaltulose, trehalulose or chitobiose. The saccharide may be a polysaccharide such as dextrin, glycogen, starch, cellulose, hemicellulose, polydextrose, inulin, β -glucan, pectin, psyllium husk mucilage, β -mannan, glucomannan, arabinoxylan, argra, alginate, carrageenan, chitin, chitosan, or various gums. The saccharide may be the D-isomer, the L-isomer, or a mixture thereof. The sugar may be a methylated derivative, an acetate derivative, a phosphate derivative or a sulfate derivative of another sugar. The composition may comprise a mixture of two or more sugars in any proportion. Other highly water soluble polymers (e.g., polyethylene glycol, polylactic acid, etc.) may also be used to make the film translucent to transparent.

The amount of sugar required to reduce autofluorescence and light scattering may depend on the thickness of the membrane immersed in the solution, as well as the excitation and emission wavelengths of the relevant fluorophores. Typically, it was found that about 70% to 100% or more weight/volume of sugar in the solution rendered the submerged film appear transparent (translucent) to nearly translucent (translucent). The amount of sugar in the composition can be 50% to 80%, 60% to 90%, 70% to 100%, 80% to 110%, or 90% to 120% weight/volume. The amount of sugar in the composition can be 70% to 82%, 72% to 84%, 74% to 86%, 76% to 88%, or 78% to 90% weight/volume. In some embodiments, the amount of sugar in the composition is 70% to 90% weight/volume. In some embodiments, the sugar concentration is 75% to 90% weight/volume. In some embodiments, the sugar concentration is about 70%, about 85%, or about 90% weight/volume.

The addition of sugar to water or buffer solution can cause the film immersed in the solution to appear transparent to translucent. However, the addition of a sugar also leads to quenching of the fluorescent signal. To reduce quenching, a surfactant may also be added to the composition. The surfactant may be anionic, cationic, nonionic, or amphoteric. In some embodiments, the surfactant is a zwitterionic surfactant. The zwitterionic surfactant can be, for example, a sulfobetaine (sultaine), a betaine, a phospholipid, or a sphingolipid. The zwitterionic surfactant may be Sulphobetaine (SB), Aminothiobetaine (ABS), or octylbenzylaminothiobetaine. The zwitterionic surfactant may be, for example, 3- ((3-cholamidopropyl) dimethylammonium) -1-propanesulfonate (CHAPS), cocamidopropyl hydroxysultaine, cocamidopropyl betaine, phosphatidylserine, phosphatidylethanolamine, phosphatidylcholine, or sphingomyelin. The zwitterionic surfactant may be an aminothiobetaine or an aromatic aminothiobetaine. In some embodiments, the zwitterionic surfactant is 3- (dimethyl (3- (4-octylbenzamido) propyl) ammonio) propane-1-sulfonate (ASB-C80). In one aspect, the surfactant is selected from: SB 3-10, SB 3-12, SB 3-14, ASB-16, or ASB-C80. The composition may comprise a mixture of two or more surfactants in any proportion.

The amount of surfactant required to reduce fluorescence quenching by the sugar may depend on the type and concentration of the sugar in solution, as well as the excitation and emission wavelengths of the relevant fluorophore. Typically, surfactants in the solution at about 0.01% to 0.2% or higher volume/volume have been found to reduce fluorescence quenching. The amount of surfactant in the composition may be 0.001% to 0.01%, 0.002% to 0.2%, 0.005% to 0.4%, 0.01% to 0.9%, or 0.02% to 2% volume/volume. The amount of surfactant in the composition can be 0.01% to 0.06%, 0.015% to 0.08%, 0.02% to 0.1%, 0.025% to 0.15%, or 0.035% to 0.02% volume/volume. In some embodiments, the concentration of the surfactant is about 0.05%, about 0.1%, or about 0.2% v/v.

The sugar and surfactant may be dissolved in water or any aqueous buffer suitable for use in an analyte detection assay. For example, the buffer may include: 2-amino-2- (hydroxymethyl) propane-1, 3-diol (Tris), Tris-HCl, Tris Buffered Saline (TBS), glycine, 2- (4- (2-hydroxyethyl) piperazin-1-yl) ethanesulfonic acid (HEPES), Phosphate Buffered Saline (PBS), or combinations thereof in any proportion. The analyte detection assay may be a Western blot. The composition can also include a membrane configured for an analyte detection assay, wherein the membrane is immersed in water or an aqueous buffer. The membrane may include: for example, nitrocellulose, polyvinylidene fluoride, nylon, or a combination of two or more thereof in any proportion.

Method III

Various methods for imaging the membrane in an analyte detection assay are also provided. The method may include: the membrane was immersed in the solution. The membrane may be configured for use in an analyte detection assay, as described above. In some embodiments, the analyte detection assay is a Western blot and the membrane comprises nitrocellulose or PVDF. The solution may have any of the compositions described above. In some embodiments, the solution comprises a sugar that is sucrose, glucose, maltose, lactose, dextrose, cellobiose, or galactose. In some embodiments, the concentration of sugar in the solution is 70% to 90% weight/volume. In some embodiments, the solution comprises a zwitterionic surfactant. The zwitterionic surfactant may be an aminothiobetaine, such as ASB-C80. In some embodiments, the surfactant concentration in the solution is 0.01% to 0.2% v/v.

The method may further comprise: the film is imaged. Imaging may be used to detect one or more analytes in any type of sample. In some embodiments, the sample is a biological sample. In some embodiments, the sample is a chemical or physical sample, for example, water or a chemical solution or air or rock. The biological sample may be obtained from any organism, such as an animal, plant, fungus, bacteria, virus or prion, or any other organism. In some embodiments, the biological sample is from an animal, such as a mammal (e.g., a human or non-human primate, cow, horse, pig, sheep, cat, dog, mouse, or rat), a bird (e.g., a chicken), or a fish. The biological sample can be any tissue or body fluid obtained from an organism, such as blood, blood components or blood products (e.g., serum, plasma, platelets, red blood cells, etc.), sputum or saliva, tissue (e.g., kidney, lung, liver, heart, brain, neural tissue, thyroid, eye, skeletal muscle, cartilage, or bone tissue); cultured cells, such as primary cultures, explants, transformed cells, stem cells, feces or urine.

In some embodiments, the one or more analytes to be detected comprise a peptide, a protein (such as an antibody, an enzyme, a growth regulator, a clotting factor, or a phosphoprotein), an immunogen, a polysaccharide, a toxin, a cell wall, a cell capsule, a viral coat, a flagella, a cilium or pilus, a microorganism, a nucleic acid complexed with a protein or polysaccharide, or a lipid complexed with a protein or polysaccharide. In some embodiments, two, three, four, five or more different analytes will be detected. In some embodiments, where two or more different analytes are to be detected, the two or more different analytes are the same type of analyte (e.g., two or more proteins present in a complex). In some embodiments, wherein two or more different analytes are to be detected, the two or more different analytes are different types of analytes. The presently described methods and compositions can be used as stand-alone single analyte assays, in which a single analyte is detected in different samples. Alternatively, the methods and compositions can be used to detect multiple analytes on many different samples separated on a transfer membrane.

The analyte may be detected using a binding agent. Binding agents described herein that are suitable for use in the methods according to the invention are any molecules that specifically bind to the analyte of interest (e.g., an antigen). In some embodiments, the binding agent is an antibody or portion thereof. In some embodiments, a binding agent described herein is linked to a detectable label. The label may be directly attached to the binding agent (e.g., by a covalent bond) or may be indirectly attached (e.g., using a chelator or linker molecule). The terms "label" and "detectable label" are used interchangeably herein and are described in detail below.

Examples of detectable labels include, but are not limited to: biotin/streptavidin labels, nucleic acid (e.g., oligonucleotide) labels, chemically reactive labels, fluorescent labels, enzymatic labels, radioactive labels, quantum dots, polymer dots, mass labels, and combinations thereof. In some embodiments, the marker may include an optical agent, such as a fluorescent agent, phosphorescent agent, chemiluminescent agent, or the like. A variety of reagents (e.g., dyes, probes, or indicators) are known in the art and can be used in the present invention. (see, e.g., Invitrogen, A manual-guidance on Fluorescent Probes and Labeling techniques (The Handbook-A Guide to Fluorescent Probes and Labeling Technologies), 10 th edition (2005)). Fluorescers may include a variety of organic and/or inorganic small molecules or a variety of fluorescent proteins and derivatives thereof. Literature sources of fluorophores include Cardullo et al (1988) Proc. Natl. Acad. Sci. USA 85:8790-8794 (1988); dexter (1953) journal of physico-Chemical (J.of Chemical Physics)21:836-850 (1953); hochstrasser et al (1992) Biophysico-Chemistry (Biophysical Chemistry)45:133-141 (1992); selvin (1995) Methods in Enzymology (Methods in Enzymology)246:300-334 (1995); steinberg (1971) Biochemical review Ann. Rev. biochem. (1971) 40: 83-114; stryer (1978) Biochemical review Ann. Rev. biochem. 47:819-846 (1978); wang et al (1990) Tetrahedron Letters 31: 6493-; and Wang et al (1995) analytical chemistry (anal. chem.)67:1197-1203 (1995). Fluorescent dyes and fluorescent marker reagents include those available from Invitrogen/Molecular Probes (Invitrogen/Molecular Probes) (ewing, oregon) and Pierce Biotechnology (Pierce Biotechnology, Inc.) and Pierce Biotechnology (rockford, illinois).

The following are non-limiting examples of fluorophores that can be used as labels: 4-acetamido-4 ' -isothiocyanatostilbene-2, 2' -disulphonic acid, acridine isothiocyanate, 5- (2' -aminoethyl) aminonaphthalene-1-sulphonic acid (EDANS), 4-amino-N- [ 3-vinylsulphonyl) phenyl]Naphthalimide-3, 5-disulfonate, N- (4-anilino-1-naphthyl) maleimide, anthranilamide, BODIPY, brilliant yellow, coumarin, 7-amino-4-methylcoumarin (AMC, coumarin 120), 7-amino-4-trifluoromethylcoumarin (coumarin 151), cyanine dyes, phloxine, 4', 6-diamidino-2-phenylindole (DAPI), 5' -dibromopyrogallol-sulfonphthalein (bromopyrogallol red), 7-diethylamino-3- (4 '-isothiocyanatophenyl) -4-methylcoumarin divinyltriamine pentaacetate, 4' -diisothiocyanatodihydro-stilbene-2, 2' -disulfonic acid, 4' -diisothiocyanatostilbene-2, 2' -disulfonic acid, 5- [ dimethylamino group]Naphthalene-1-sulfonyl chloride (DNS, dansyl chloride), 4- (4 '-dimethylaminophenylazo) benzoic acid (DABCYL), 4-dimethylaminophenylazophenyl-4' -isothiocyanate (DABITC), eosin isothiocyanate, erythrosine B, erythrosine isothiocyanate, ethidine, 5-carboxyfluorescein (FAM), 5- (4, 6-dichlorotriazin-2-yl) aminofluorescein (DTAF), 2',7' -dimethoxy-4 ',5' -dichloro-6-carboxyfluorescein (JOE), fluorescein isothiocyanate, fluoramine, IR144, IR1446, malachite green isothiocyanate, 4-methylumbelliferone, o-cresolphthalein, nitrotyrosine, parafuchsin, phenol red, phycoerythrin (including but not limited to B-type and R-type), Ortho-phthalaldehyde, pyrene butyrate and amberPerimido 1-pyrene butyrate, quantum dots and reactive red 4 (CIBACRON)^TMBrilliant red 3B-a), 6-carboxy-X-Rhodamine (ROX), 6-carboxy rhodamine (R6G), lissamine rhodamine B sulfonylchloride rhodamine (Rhod), rhodamine B, rhodamine 123, rhodamine X isothiocyanate, sulforhodamine B, sulforhodamine 101, sulfonyl chloride derivatives of sulforhodamine 101 (texas red), N' -tetramethyl-6-carboxy rhodamine (TAMRA), Tetramethyl Rhodamine Isothiocyanate (TRITC), riboflavin, rhodizonic acid, lanthanide chelate derivatives. In some embodiments, the optical agent is an intercalating dye. Intercalating dyes include, but are not limited to, SYBR Green and Pico Green (from Molecular Probes, Europe, Oregon)), ethidium bromide, propidium iodide, chromomycin, acridine orange, Hoechst 33258, TOTO-I, YOYO-1, and DAPI (4', 6-diamidino-2-phenylindole hydrochloride).

A preferred group of fluorophores for use in immunoassays includes fluorescein, fluorescein isothiocyanate, phycoerythrin, rhodamine B, and texas red (sulfonyl chloride derivative of sulforhodamine 101). Any of the fluorophores listed in the previous list of this paragraph can be used in the assays described herein for labeling a microparticle or labeling a binding agent (e.g., an antibody or streptavidin). The fluorescent dye may be attached by conventional covalent attachment using appropriate functional groups on the fluorophore and on the microparticle or binder. The identification of such groups and the reactions to form the linkages will be apparent to those skilled in the art.

In some embodiments, the fluorescent agent is a polymer dot or a quantum dot. The particular Quantum Dots (QDs) used are not important to the present invention. Quantum dots are known in the art and described, for example, in Han et al, "Quantum-dot-tagged Microbeads for Multiplexed Optical Coding of Biomolecules" (Nat Biotechnol (2001, 7) Vol.19, p.631-635). One skilled in the art will appreciate that there are a variety of quantum dots that can be used as fluorescent labels and for embodiments of the present invention and are available from a variety of vendors. Exemplary quantum dots (ODs) include, but are not limited to, the following: amount of cadmium selenide (CdSe)Sub-dot nanoparticles (e.g., CdSe quantum dot core, 480-640nm emission spectrum, Sigma-Aldrich Co.)

) (ii) a Cadmium sulfide (CdS) quantum dot nanoparticles (e.g., CdS quantum dot core, 380-; zinc sulfide capped cadmium selenide (ZnS capped CdSe) nanocrystals (e.g., CdSe/ZnS LUMIDOTS)^TMAnd CdSe/ZnS NanODOTS^TM480-; and cadmium-free quantum dots (e.g., CFQDs)^TM400-650nm emission spectrum, Sigma-Aldrich).

Techniques for attaching detectable labels to binding agents are known. For example, a review of common protein labeling techniques can be found in biochemical techniques: theory and Practice (Biochemical technologies: the order and Practice), John F.Robyt and Bernard J.white, Viverland publishing Co., Waveland Press, Inc. (1987). Other labeling techniques are reviewed, for example, in "excitation States of Biopolymers (R. Haughland, exposed States of Biopolymers)," Steiner, Pluronic Press (1983); fluorescent probe design and synthesis: technical Guide (fluorine Probe Design and Synthesis: A Technical Guide), PE Applied Biosystems (PE Applied Biosystems) (1996); and g.t.herman, "Bioconjugate Techniques," Academic Press (1996). The technique may also be performed as a commercially available kit (e.g., Innova Biosciences Ltd., Cambridge, England)

And

) Is obtained in part. Many appropriately labeled binding agents are commercially available and may be used with or without further modification. Other labels that can be used in place of fluorophores are radioactive labels and enzyme labels. These methods are also in the artAre well known.

IV. reagent kit

Also provided are a plurality of kits for detecting one or more analytes. The analyte may be any analyte as described above. In some embodiments, the analyte is a protein. The kit comprises a sugar and a surfactant. The sugar may comprise any one or more of the sugars described above. The surfactant may comprise any one or more of the surfactants described above. In some embodiments, the saccharide is selected from the group consisting of: sucrose, glucose, maltose, lactose, dextrose, cellobiose, and galactose; and the surfactant is an aminothiobetaine. The kit may also comprise a buffering agent. The buffer may include any one or more of the buffer compounds described above. In some embodiments, the sugar, surfactant and/or buffer are provided as dry ingredients, combined into an aqueous solution by the end user.

The kit may further comprise a membrane. The membrane may be any of the membranes described above. In some embodiments, the membrane is configured for use in an analyte detection assay. In some embodiments, the membrane is configured for use in a Western blot assay. In some embodiments, the membrane comprises nitrocellulose or PVDF.

The kit may further comprise a binding agent. The binding agent may comprise any one or more of the binding agents described above. For example, as described herein, a kit can comprise one or more antibodies labeled with biotin, and/or an analyte-specific antibody pair, (strept) avidin, labeled biotin, e.g., phycoerythrin-labeled biotin, beads, control reagents. In some embodiments, the kit further comprises instructions for performing the methods described herein.

V. examples

Example 1 Effect of sugar solution on transparency of submerged membranes

Both PVDF and nitrocellulose membranes scatter a lot of light in air and water due to the porous nature of the solid and the large difference in refractive index between the membrane (>1.45) and either air (1.00) or water (about 1.33). If the refractive index between the solid and the environment is matched, the light no longer has a surface to scatter and the film appears almost transparent. This can be produced by adding and further controlling the amount of sugar in the solution in which the membrane is immersed.

Two similar PVDF membranes were immersed in 85% sugar solution and water (fig. 1). As can be seen, the left PVDF membrane immersed in the sugar solution becomes almost transparent. Such a clearly transparent film has a significantly reduced background signal when imaged with, for example, a ChemiDoc MP imaging system (Bio-Rad).

Example 2 Effect of surfactants on fluorescence quenching by sugars

Sugars are polar molecules, and polar solvents typically quench fluorophores. FIG. 2 shows the emission spectrum of the fluorophore in aqueous sugar water. According to the data in the figure, the emission intensity in neutral phosphate buffer decreased by about 50% after addition of 85% w/v sucrose. However, further addition of surfactant (0.1% ASB-C80) to the sugar buffer solution reduced this sugar-induced quenching, thereby restoring about half the lost intensity. In addition, the data in FIG. 3 shows that the addition of surfactant (0.1% ASB-C80) to phosphate or other standard buffers also increases the fluorescence signal from the buffer.

Example 3 fluorescence intensity of imprinted proteins imaged with sugar and surfactant solutions

Figure 4 shows the signal intensity of the protein detected on the membrane soaked in sugar water compared to the intensity of the protein detected on the membrane soaked in sugar water + surfactant. Both films were imaged simultaneously with the same exposure. The results shown indicate that membranes soaked in sugar and surfactant produce greater signal intensity, which is consistent with the fluorescence emission results of fig. 2 and 3.

Example 4 imaging of cell lysates Using sugar and surfactant solutions

Samples of the isolated cell lysates were blotted onto PVDF membranes and then imaged in the presence and absence of an aqueous buffer comprising a sugar and a surfactant. The image presented in fig. 5 shows no dye emission from the sample, and it can be seen that the resolution of the protein bands within the sample is enhanced when imaged using the sugar/surfactant buffer. As can be seen from the graph in fig. 6, the reduction in light scattering and the increase in emission intensity appear to be consistent with the resolution enhancement in the case of imaging in the presence of sugars and surfactants.

Example 5 Effect of surfactant type and concentration

Four different zwitterionic surfactants were tested for their efficiency in reducing fluorescence quenching by sugar solutions. From the graph shown in fig. 7, among these surfactants, the aromatic aminosulfobetaine ASB-C80 exhibited the best ability to mitigate quenching and produced an image with the greatest fluorescence intensity. Further experiments were conducted to determine the concentration effect of ASB-C80 on reducing fluorescence quenching. As shown in fig. 8, all concentrations tested exhibited sufficient reduction in quenching.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of the appended claims. In addition, each reference provided herein is incorporated by reference in its entirety as if each reference were individually incorporated by reference. Instant applications dominate when there is a conflict between the instant application and the references provided herein.

Claims

1. A composition comprising a film immersed in a solution comprising:

70% w/v or more sugar; and

a zwitterionic surfactant, wherein the sugar and zwitterionic surfactant are dissolved in water or an aqueous buffer; and is

The membrane is configured for use in an analyte detection assay.

2. The composition of claim 1, wherein the membrane comprises nitrocellulose or polyvinylidene fluoride (PVDF).

3. The composition of any one of claims 1-2, wherein the zwitterionic surfactant is an aminothiobetaine.

4. The composition of claim 3, wherein said aminothiobetaine is 3- (dimethyl (3- (4-octylbenzamido) propyl) ammonio) propane-1-sulfonate (ASB-C80).

5. The composition of claim 1, wherein the sugar is selected from the group consisting of: sucrose, glucose, maltose, lactose, dextrose, cellobiose, and galactose.

6. The composition of claim 1, wherein the sugar concentration is in the range of 70% to 90% w/v.

7. The composition of claim 1, wherein the zwitterionic surfactant concentration is in the range of 0.01% to 0.2% v/v.

8. A method of imaging a membrane in an analyte detection assay, the method comprising:

immersing a membrane in a solution, wherein the membrane is configured for an analyte detection assay, wherein the solution comprises 70% w/v or more of a sugar and a zwitterionic surfactant, and wherein the sugar and zwitterionic surfactant are dissolved in water or an aqueous buffer; and

imaging the film.

9. The method of claim 8, wherein the membrane comprises nitrocellulose or polyvinylidene fluoride (PVDF).

10. The method of claim 8 or 9, wherein the zwitterionic surfactant is an aminothiobetaine.

11. The method of claim 10, wherein the aminothiobetaine is 3- (dimethyl (3- (4-octylbenzamido) propyl) ammonio) propane-1-sulfonate (ASB-C80).

12. The method of claim 8, wherein the sugar is selected from the group consisting of: sucrose, glucose, maltose, lactose, dextrose, cellobiose, and galactose.

13. The method of claim 8, wherein the sugar concentration is in the range of 70% to 90% w/v.

14. The method of claim 8, wherein the zwitterionic surfactant concentration is in the range of 0.01% to 0.2% v/v.

15. A kit for detecting an analyte, the kit comprising:

a solution, the solution comprising:

at least 70% w/v or more of a sugar;

a zwitterionic surfactant; and

a buffering agent; and

a membrane configured for use in an analyte detection assay.

16. The kit of claim 15, wherein the zwitterionic surfactant is aminothiobetaine.

17. The kit of claim 16, wherein said aminothiobetaine is 3- (dimethyl (3- (4-octylbenzamido) propyl) ammonio) propane-1-sulfonate (ASB-C80).

18. The kit of any one of claims 15 to 17, wherein the saccharide is selected from the group consisting of: sucrose, glucose, maltose, lactose, dextrose, cellobiose, and galactose.

19. The kit of claim 15, further comprising:

a binding agent.